Foetal Imaging: Optimising Lugol’s Solution Staining for CT Scanning

Abstract

Biomedical researchers and clinicians are interested in (ab)normal foetal development because it can aid in better understanding human anatomy. To capture this foetal development non-destructive three dimensional (3D) imaging techniques like computed tomography (CT) are used. Visualising foetuses remains a challenge however, as foetuses consist mostly of soft-tissue. Visualisation of soft-tissue with CT scans is difficult because X-rays easily pass through. This consequently results into images with low contrast. Therefore, improving contrast is artificially gained by using chemical compounds called stains. The most effective stain is considered to be Lugol’s solution. A downside of using Lugol’s solution is that the staining process causes extensive soft-tissue shrinkage which is detrimental for morphological analysis. The mechanism of Lugol-induced shrinkage is largely unknown. Some research suggest it is due to an osmotic imbalance between tissue and solution, while others point towards acidification of Lugol’s solution. The goal of this study is to develop an optimum (buffered) Lugol’s solution staining protocol for post-mortem human foetal CT imaging to diminish soft-tissue shrinkage and achieve homogeneous staining. Several variables in the protocol are taken into account such as staining solution concentration, staining time and specimen size. To develop this protocol, multiple tests and measurements (pH, osmolarity, optical density, weight and CT scans) were performed on pork liver samples and two post-mortem human foetuses to monitor acidification of the staining solution, staining progress and staining intensity, while applying two distinct methods: the AMC- and Arthurs method. The main difference between these methods is that the AMC method fixates tissue well before staining (conventional method), while Arthurs method uses a mixture of a fixative and stain simultaneously on fresh tissue. The research suggests that Arthurs method seems best. Even though, both methods led to a homogeneous staining, the AMC method resulted in an average shrinkage of 4.82%, while Arthurs method resulted in a shrinkage of only 1.08%. In addition, Arthurs method leads to a shorter staining protocol.